840:153g:Projects/project1/2008/11/11

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A Flower of A Different Color
We, vector group (Angie and Diwash) did the agarose gel electrophoresis of PCR products of pSB1A3 and pSB1A7 plasmids. We found the primer size of pSB1A3 around 350 base pair which should be 316 bp. But the primer size of pSB1A7 is around 800 bp which should be 590 bp. So, we conclude that the pSB1A7 is not our target plasmid. Hence, from the result we can conclude that pSB1A3 can be used as our target plasmid where we can insert the gene of interest i.e. cytochrome p450 for the blue pigmentation. Since all the positive (pBLUE) and negative (no plasmid samples)control works well. But, there occure the faint band in the negative control which may be due to transfer of PCR products while loading on the gel.

Sushma and Binu performed a double digest of extracted plasmids pSB1A2, each with BBa_R0040, BBa_R0051 promoter to verify the promoters and plasmid identity. We used fast-digest enzymes, SpeI & XbaI to digest the 10micro liters of plasmid DNA and then ran gel for 40 minutes at 150V. we use more DNA this time so that we can see a faint band of 50bp of promoter.From the gel we can see a faint band below 100bp.But there are 3 bands around 2000,1500 and 1200 bp.This may be due to more concentration of DNA that the Restriction enzymes are unable to digest.From the gel we got an identity of plasmid PSB1A2 each with promoter BBa_R0040,BBa_R0051,BBa_R0010.

Melissa, Tiffany and Rajiv worked on RT-PCR of the RNA extraction product using the RNA product from tube 2. The reasoning for using this RNA product verse both RNA products obtained is due to the fact that RNA tube 2 had 0.42 grams of flower material used during the RNA extraction. The RT-PCR had conditions tested during PCR using one tube labeled with a green dot for no reverse transcription and kept on ice while the tube with no labeling was heated during the Reverse transcription in the thermocycler. The product will be stored in -20C till the next lab time. The protocol for RT-PCR was from the Qiagen One Step RT-PCR Kit hand book 05/2002 [1]. The RT-PCR kit is near to expiration date which is 12/2008 this may cause problems with the RT-PCR preformed today. The results for the DNA PCR problems are not due to the PCR master mix being to old or damaged. The problem has to be with PCR setup and actual extracted DNA being tested, DNA PCR will be completed during the next lab meeting.